Effects And Mechanism Of Oxalated Zero-Valent Iron Catalytic Ozonation To Remove Antibiotic Resistance

Antibiotic resistance refers to some microorganisms’characteristic of high tolerance to antibiotics,that spread in the environment mainly through antibiotic resistance bacteria（ARB）and antibiotic resistance genes（ARGs）,posing a significant threat to human health.Ozone disinfection can remove ARB and ARGs in sewage to some extent,but due to the limited oxidation ability,the incompletely degraded ARB and ARGs can be repaired under suitable conditions then continue to spread.Zero-valent iron（ZVI）can promote ozonation to produce hydroxyl radicals with higher oxidation capacity,which can improve the removal rate of ARB and ARGs.However,ZVI suffers from the low reactivity of catalyzing ozonation because of generating passive iron oxide shell on the surface,our group previously proposed a method for modifying ZVI with oxalic acid,the prepared oxalated zero-valent iron(OA-ZVI^bm)greatly improved the capacity of catalyzing ozonation.But till now,there is few report on OA-ZVI^bmcatalytic ozonation to simultaneously remove ARB and ARGs in water.Herein,in this study,the double-resistant bacterium Escherichia coli（AR E.coli）carrying resistance genes tetC and blaTEM was prepared,and OA-ZVI^bmwas prepared by mechanical ball milling.We used scanning electron microscopy（SEM）,X-ray diffractometer（XRD）and Fourier transform infrared spectroscopy（FT-IR）to characterize the surface properties of OA-ZVI^bmsamples;the effect of inactivation of AR E.coli and degradation of tetC and blaTEM was detected by plate counting and fluorescence quantitative PCR techniques.In order to explore the mechanism of OA-ZVI^bmcatalytic ozonation,pH value and Fe²⁺concentration of the system was analyzed,the reactive oxygen species（ROSs）were detected by electron paramagnetic resonance（EPR）and fluorescence spectrophotometer,and the quenching tests were implemented to investigate the contribution of the generated various ROSs in AR E.coli inactivation.Further,the pathway of ARE.coli inactivation was explored by membrane damage detection and intracellular ROS content detection;the risk of horizontal gene transfer was analyzed by agarose gel electrophoresis and transformation assay.Research results are as follows:（1）SEM images showed that OA-ZVI^bmparticles had a rough surface,composed of a large number of irregular sheet structures with voids;the XRD pattern showed that OA-ZVI^bmhad characteristic peak ofα-Fe⁰at 44.7°and 65°;Fourier transform infrared spectroscopy spectrum showed that the characteristic peaks of OA-ZVI^bmsamples were basically consistent with that of the control sample FeC₂O₄·2H₂O.The above characterization results confirmed that OA-ZVI^bmwith FeC₂O₄·2H₂O layer was successfully prepared by mechanical ball milling technology.（2）The results of plate counting showed that the inactivation effect of AR E.coli by400 mg/L OA-ZVI^bm（the molar ratio of oxalic acid and ZVI was 2%）coupled with ozone of 20 mL/min was more significant.Under this condition,the concentration of AR E.coli decreased by 2.62,3.82,6.58 and 6.58 log when treated for 15,30,45 and 60 min,respectively,and the inactivation rate of AR E.coli reached 100%after 45 min.Its sterilization effect is significantly higher than that of O₃（0.20,1.07,2.87 and 4.64 log reduction for the above four time points）and ZVI^bm/O₃treatment group（1.48,3.00,3.49and 6.58 log reduction for the above four time points）.The results of real-time PCR showed that the degradation effect of OA-ZVI^bm/O₃on tetC and blaTEM（2.49 and 2.83log reduction of tetC and blaTEM after 60 min）was significantly better than that of O₃（0.53 and 0.66 log reduction of tetC and blaTEM）and ZVI^bm/O₃（1.20 and 1.54 log reduction of tetC and blaTEM）system.（3）The pH value of the control system(O₃and ZVI^bm/O₃)decreased gradually with the extension of reaction time,while that of OA-ZVI^bm/O₃system did not decrease significantly.The colorimetry of complex and 1,10 phenanthrolin result showed that OA-ZVI^bm/O₃system dissolved more Fe²⁺than ZVI^bm/O₃system.The results above suggested that the shell structure of OA-ZVI^bmaccelerated the hydrogen evolution reaction between iron core and water to produce more Fe²⁺.After capturing Fe（Ⅱ）,the inactivation rate of ZVI^bm/O₃system and OA-ZVI^bm/O₃system against AR E.coli decreased by 45.6%and 87.3%,respectively,which suggested that Fe（Ⅱ）played an important role in the inactivation of AR E.coli by catalytic ozonation.Further,Fe²⁺catalytic ozonation(Fe²⁺/O₃)system was constructed by ferrous sulfate to inactivate AR E.coli.The results showed that,removal rates of AR E.coli in O₃,Fe²⁺/O₃and OA-ZVI^bm/O₃systems were 1.07,1.10 and 5.05 log values,respectively,which indicated that solution phase Fe²⁺couldn’t catalyze ozonation,and the effective component of OA-ZVI^bmcatalytic ozonation was Fe（Ⅱ）adsorbed on the catalyst surface.（4）The results of electron paramagnetic resonance and fluorescence spectrophotometer showed that hydroxyl radicals（·OH）,superoxide anions（·O₂^–）and singlet oxygen（¹O₂）were generated in the OA-ZVI^bm/O₃system.After capturing·OH and ¹O₂,the inactivation rate of AR E.coli in OA-ZVI^bm/O₃system decreased by 83.7%and 88.7%,respectively;while the capture of·O₂^–had almost no effect on the inactivation of AR E.coli.The results above suggested that·OH and ¹O₂produced by OA-ZVI^bm/O₃system were the main active species in inactivating AR E.coli.（5）Membrane damage detection results showed that the cell membrane of AR E.coli changed from smooth to rough,with wrinkles and holes,and degraded gradually to envelope debris.The results of intracellular ROS levels showed that the intracellular ROS level in OA-ZVI^bm/O₃treatment group increased sharply,reached a peak at 30 min and then decreased due to the decrease of bacteria abundance.The results of agarose gel electrophoresis showed that the brightness of the electrophoresis bands of blaTEM and tetC decreased significantly after 15 min of OA-ZVI^bm/O₃treatment,and further darkened with time,indicating that OA-ZVI^bm/O₃system degraded ARGs with high efficiency.The results of transformation experiments showed that OA-ZVI^bm/O₃treatment for 15 min could effectively prevent horizontal gene transfer of extracellular ARGs.In conclusion,Fe（Ⅱ）on oxalated zero-valent iron surface could efficiently catalyze ozonation to produce a large number of reactive oxygen species such as·OH and ¹O₂,which could effectively remove ARB and ARGs and inhibit horizontal gene transfer.This study provided a new research idea and theoretical support for the effective removal of resistant bacteria and resistance genes.